PROJECT SUMMARY Eccrine glands are the major appendage of human skin and required for human thermoregulation. Despite their importance, the cellular and molecular mechanisms of their development are poorly understood. This proposal builds on our work implicating the transcription factor Engrailed-1 as a unique determinant and driver of eccrine gland formation to define genetic and cellular mechanisms that induce the specification and differentiation of eccrine sweat glands in the skin. Accordingly, the goals of this proposal are to identify the transcription factors that directly modulate Engrailed1 expression during eccrine gland development (Aim1) and to define the downstream Engrailed 1-dependent transcriptional program that then executes eccrine gland formation (Aim2). This proposal integrates in vivo testing and high-throughput discovery of cellular and molecular components of eccrine gland development using both newly generated and existing mouse models combined with mechanistic dissection in cultured human skin cells and cross-validation between novel mouse and human single nucleus transcriptomic datasets. As such, the proposed experiments will delineate the eccrine gland developmental program, inform efforts to establish an in vitro system in which to study eccrine glands, and enhance understanding of how ectodermal appendage fate and formation are established in the skin more broadly. From a clinical perspective, our findings will provide a molecular blueprint for future studies to regenerate eccrine glands in vivo and also offer novel points of intervention for repair of eccrine glands when these organs are pathologically altered or damaged.